PROJECT SUMMARY/ABSTRACT The overall goal of our continuing research program is to develop advanced technologies based on electrospray ionization (ESI) with mass spectrometry (MS) to support structural biology efforts. The role of protein assemblies in normal cellular processes and diseases warrants a practical and sensitive method for their study. The structural determination of protein complexes can play an important role in the fundamental understanding of biochemical pathways. New techniques based on native mass spectrometry (i.e., the measurement of biomolecules in their native solution environment to preserve interactions with ligands and other molecules) will be advanced to facilitate the characterization of protein assemblies. Improved methods for measuring large protein complexes (including >0.5 MDa) will be developed. Native protein separations for subsequent analysis by native MS will be advanced. Experimental MS protocols for difficult-to-characterize membrane proteins will be developed. Enhancing multiple charging generated by ESI increases the efficiency for top-down MS (the direct fragmentation of intact gas-phase large molecules to generate structure-informative product ions) and native MS; we will identify new charge enhancing agents, and we will apply these new reagents for top-down MS and membrane protein MS. With high-resolution Fourier transform-ion cyclotron resonance mass spectrometry, enhanced native top-down MS methods will be developed to obtain structurally-relevant information for large protein complexes. Methods incorporating UV photodissociation, electron capture dissociation, and electron ionization dissociation will be developed and tested for their efficiency to generate sequence information from large protein complexes and membrane proteins. Strategies for determining collision cross sections will be improved. These advanced tools will be applied to characterize complexes of biological importance where high-resolution structures are unavailable, including G-coupled protein receptors (GPCRs). Our experimental strategies are broadly applicable to be integrated with different types of biophysical techniques; such integration will allow the study of large and complex molecular machines in greater detail, providing insight into the functional dynamics of the system. Native top-down MS will be a promising approach to advance structural biology and hasten drug discovery and development.